Protein Labeling – Improved quantitation of biomolecular interactions by MicroScale Thermophoresis using the RED-NHS 2nd Generation labeling kit

MicroScale Thermophoresis (MST) is a biophysical technique that measures the strength of the interaction between two molecules by detecting variations in fluorescence signal as a result of an IR-laser induced temperature change. TRIC (Temperature Related Intensity Change) together with thermophoresis contribute to the variation of fluorescence measured. TRIC is an effect where the fluorescence intensity of a fluorophore is dependent upon temperature changes and binding interactions. Thermophoresis is the quantifiable movement of fluorescent molecules along a temperature gradient.

With the goal of improving the quality and robustness of MST measurements we selected fluorophores that are more sensitive to temperature changes and are therefore better reporters of the binding interaction. We show that the use of a TRIC-optimized Monolith Protein Labeling Kit RED-NHS 2nd Generation boosts the sensitivity of MST measurements, resulting in improved performance compared to a previously released kit. In binding affinity experiments the new reagents yield vastly improved signal-to-noise ratios and binding amplitudes, ensuing higher quality binding data and in some cases where other fluorophores tested failed, making binding affinity measurement possible. The kit is offered for our Monolith systems with red filtersets.